Accelerated Protein Degradation Research Articles

Phytochrome-interacting factors PIF4 and PIF5 directly regulate autophagy during leaf senescence in Arabidopsis.

During leaf senescence, autophagy plays a critical role by removing damaged cellular components and participating in nutrient remobilization to sink organs. However, how AUTOPHGAY (ATG) genes are regulated during natural leaf senescence remains largely unknown. In this study, we attempted to identify upstream transcriptional regulator(s) of ATGs and their molecular basis during leaf senescence in Arabidopsis through the combined analyses of promoter binding, autophagy flux, and genetic interactions. We found that PIF4 and PIF5 (PIF4/PIF5) directly bind to the promoters of the ATG5, ATG12a, ATG12b, ATG8a, ATG8e, ATG8f, and ATG8g, inducing their transcription. These target ATGs are downregulated in pif4, pif5, and pif4pif5 mutants, resulting in decreased autophagic activity and slower degradation of chloroplast proteins and chlorophyll. Conversely, overexpression of ATG8s accelerated protein degradation with early leaf senescence. Moreover, our data suggests partial suppression of the pif4pif5 phenotype by ATG8a overexpression. PIF4/PIF5 also influences senescence induced by nutrient starvation, another hallmark of the autophagy pathway. Furthermore, we observed that the PIF4/PIF5-ATG regulatory module may contribute to seed maturation. Our study not only unveils transcriptional regulators of autophagy in natural leaf senescence but also underscores the potential role of PIF4/PIF5 as functional regulators in leaf senescence and nutrient remobilization.

The therapeutic potential of red beetroot (Beta vulgaris L.) intake on muscle atrophy in immobilized mouse skeletalmuscles.

Skeletal muscle atrophy is the reduction in muscle mass and function caused by an imbalance in protein synthesis and degradation. Inflammation has been shown to accelerate protein degradation during periods of muscle inactivity. We investigated the potential therapeutic effects of beetroot extract (BRE) in reducing inflammation and oxidative stress to prevent muscular atrophy after a short period of immobilization. We divided 36 male BALB/c mice into three groups: control, muscular atrophy mice, and muscular atrophy mice treated with BRE (n = 12). Each group was further divided into two subgroups: (1) 7-day immobilization and (2) 10-day recovery. BRE was administered orally at a dose of 300 mg/kg for 17 days. We assessed the anti-inflammatory and antioxidative effects of BRE using ELISA and RT-PCR assays. Hematoxylin-eosin staining was used to measure the cross-sectional area (CSA) of muscle fibers, and grip strength tests were performed to assess muscle strength. BRE administration increased muscle weight, CSA, and grip strength in mice with immobilization-induced muscle atrophy. It also suppressed inflammation and oxidative stress biomarkers in atrophic muscle fibers. Higher nitrate levels and lower Troponin I (TnI) concentrations were observed in the BRE-treated group, indicating improved muscle function and structure. These findings suggest that BRE may have therapeutic benefits in improving muscle mass and function and warrant further studies in humans, particularly in individuals with low physical activity levels.

A Homozygous Variant in NAA60 Is Associated with Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a monogenic disorder characterized by bilateral calcifications in the brain. The genetic basis remains unknown in over half of the PFBC patients, indicating the existence of additional novel causative genes. NAA60 was a recently reported novel causative gene for PFBC. The aim was to identify the probable novel causative gene in an autosomal recessive inherited PFBC family. We performed a comprehensive genetic study on a consanguineous Chinese family with 3 siblings diagnosed with PFBC. We evaluated the effect of the variant in a probable novel causative gene on the protein level using Western blot, immunofluorescence, and coimmunoprecipitation. Possible downstream pathogenic mechanisms were further explored in gene knockout (KO) cell lines and animal models. We identified a PFBC co-segregated homozygous variant of c.460_461del (p.D154Lfs*113) in NAA60. Functional assays showed that this variant disrupts NAA60 protein localization to Golgi and accelerated protein degradation. The mutant NAA60 protein alters its interaction with the PFBC-related proteins PiT2 and XPR1, affecting intracellular phosphate homeostasis. Further mass spectrometry analysis in NAA60 KO cell lines revealed decreased expression of multiple brain calcification-associated proteins, including reduced folate carrier (RFC), a folate metabolism-related protein. Our study replicated the identification of NAA60 as a novel causative gene for autosomal recessive PFBC, demonstrating our causative variant leads to NAA60 loss of function. The NAA60 loss of function disrupts not only PFBC-related proteins (eg, PiT2 and XPR1) but also a wide range of other brain calcification-associated membrane protein substrates (eg, RFC), and provided a novel probable pathogenic mechanism for PFBC. © 2024 International Parkinson and Movement Disorder Society.

Heterozygosity for loss-of-function variants in LZTR1 is associated with isolated multiple café-au-lait macules

PurposePathogenic LZTR1 variants cause schwannomatosis and dominant/recessive Noonan syndrome (NS). We aim to establish an association between heterozygous loss-of-function LZTR1 alleles and isolated multiple café-au-lait macules (CaLMs). MethodsA total of 849 unrelated participants with multiple CaLMs, lacking pathogenic/likely pathogenic NF1 and SPRED1 variants, underwent RASopathy gene panel sequencing. Data on 125 individuals with heterozygous LZTR1 variants were collected for characterizing their clinical features and the associated molecular spectrum. In vitro functional assessment was performed on a representative panel of missense variants and small in-frame deletions. ResultsAnalysis revealed heterozygous LZTR1 variants in 6.0% (51/849) of participants, exceeding the general population prevalence. LZTR1-related CaLMs varied in number, displayed sharp or irregular borders, and were generally isolated but occasionally associated with features recurring in RASopathies. In 2 families, CaLMs and schwannomas co-occurred. The molecular spectrum mainly consisted of truncating variants, indicating loss-of-function. These variants substantially overlapped with those occurring in schwannomatosis and recessive NS. Functional characterization showed accelerated protein degradation or mislocalization, and failure to downregulate mitogen-activated protein kinase signaling. ConclusionOur findings expand the phenotypic variability associated with LZTR1 variants, which, in addition to conferring susceptibility to schwannomatosis and causing dominant and recessive NS, occur in individuals with isolated multiple CaLMs.

Transcriptome Analysis of miRNA and mRNA in Porcine Skeletal Muscle following Glaesserella parasuis Challenge.

Glaesserella parasuis (G. parasuis) causes systemic infection in pigs, but its effects on skeletal muscle and underlying mechanisms are poorly understood. We investigated G. parasuis infection in colostrum-deprived piglets, observing decreased daily weight gain and upregulation of inflammatory factors in skeletal muscle. Muscle fiber area and diameter were significantly reduced in the treated group (n = 3) compared to the control group (n = 3), accompanied by increased expression of FOXO1, FBXO32, TRIM63, CTSL, and BNIP3. Based on mRNA and microRNA (miRNA) sequencing, we identified 1642 differentially expressed (DE) mRNAs and 19 known DE miRNAs in skeletal muscle tissues between the two groups. We predicted target genes with opposite expression patterns to the 19 miRNAs and found significant enrichment and activation of the FoxO signaling pathway. We found that the upregulated core effectors FOXO1 and FOXO4 were targeted by downregulated ssc-miR-486, ssc-miR-370, ssc-miR-615, and ssc-miR-224. Further investigation showed that their downstream upregulated genes involved in protein degradation were also targeted by the downregulated ssc-miR-370, ssc-miR-615, ssc-miR-194a-5p, and ssc-miR-194b-5p. These findings suggest that G. parasuis infection causes skeletal muscle atrophy in piglets through accelerated protein degradation mediated by the "miRNAs-FOXO1/4" axis, while further research is necessary to validate the regulatory relationships. Our results provide new insights into the understanding of systemic inflammation growth mechanisms caused by G. parasuis and the role of miRNAs in bacterial infection pathogenesis.

KCTD5 regulates Ikaros degradation induced by chemotherapeutic drug etoposide in hematological cells.

Therapy-related leukemia carries a poor prognosis, and leukemia after chemotherapy is a growing risk in clinic, whose mechanism is still not well understood. Ikaros transcription factor is an important regulator in hematopoietic cells development and differentiation. In the absence of Ikaros, lymphoid cell differentiation is blocked at an extremely early stage, and myeloid cell differentiation is also significantly affected. In this work, we showed that chemotherapeutic drug etoposide reduced the protein levels of several isoforms of Ikaros including IK1, IK2 and IK4, but not IK6 or IK7, by accelerating protein degradation, in leukemic cells. To investigate the molecular mechanism of Ikaros degradation induced by etoposide, immunoprecipitation coupled with LC-MS/MS analysis was conducted to identify changes in protein interaction with Ikaros before and after etoposide treatment, which uncovered KCTD5 protein. Our further study demonstrates that KCTD5 is the key stabilizing factor of Ikaros and chemotherapeutic drug etoposide induces Ikaros protein degradation through decreasing the interaction of Ikaros with KCTD5. These results suggest that etoposide may induce leukemic transformation by downregulating Ikaros via KCTD5, and our work may provide insights to attenuate the negative impact of chemotherapy on hematopoiesis.

Myofibril degradation and structural changes in myofibrillar proteins of porcine longissimus muscles during frozen storage

The effect of frozen time and the temperature on myofibril degradation and the structure of myofibrillar proteins of porcine longissimus muscles were investigated. With extended frozen time and increased temperature, the muscle fibres became broken; the muscle cells became irregularly arranged; and the fragmentation index value, number of ionic bonds, and number of hydrogen bonds of the samples significantly decreased. Meanwhile, the myofibril fragmentation index value, number of hydrophobic interactions, and number of disulphide bonds significantly increased (P < 0.05). After 12 months of storage, the intensities of I760/I1003, I850/I830, I1450/I1003, and I2945/I1003 in the samples frozen at −8 °C were reduced by 4.36 %, 1.28 %, 1.86 %, and 0.74 %, respectively. A reduction in the maximum absorption peak and a red shift were observed in the ultraviolet spectrum. Therefore, frozen storage resulted in significant damage to the tissue microstructureand caused accelerated protein degradation, and the loss of protein structural integrity.

Ubiquitination of acetyltransferase Gcn5 contributes to fungal virulence in Fusarium graminearum.

The histone acetyltransferase general control non-depressible 5 (Gcn5) plays a critical role in the epigenetic landscape and chromatin modification for regulating a wide variety of biological events. However, the post-translational regulation of Gcn5 itself is poorly understood. Here, we found that Gcn5 was ubiquitinated and deubiquitinated by E3 ligase Tom1 and deubiquitinating enzyme Ubp14, respectively, in the important plant pathogenic fungus Fusarium graminearum. Tom1 interacted with Gcn5 in the nucleus and subsequently ubiquitinated Gcn5 mainly at K252 to accelerate protein degradation. Conversely, Ubp14 deubiquitinated Gcn5 and enhanced its stability. In the deletion mutant Δubp14, protein level of Gcn5 was significantly reduced and resulted in attenuated virulence in the fungus by affecting the mycotoxin production, autophagy process, and the penetration ability. Our findings indicate that Tom1 and Ubp14 show antagonistic functions in the control of the protein stability of Gcn5 via post-translational modification and highlight the importance of Tom1-Gcn5-Ubp14 circuit in the fungal virulence. IMPORTANCE Post-translational modification (PTM) enzymes have been reported to be involved in regulating numerous cellular processes. However, the modification of these PTM enzymes themselves is largely unknown. In this study, we found that the E3 ligase Tom1 and deubiquitinating enzyme Ubp14 contributed to the regulation of ubiquitination and deubiquitination of acetyltransferase Gcn5, respectively, in Fusarium graminearum, the causal agent of Fusarium head blight of cereals. Our findings provide deep insights into the modification of acetyltransferase Gcn5 and its dynamic regulation via ubiquitination and deubiquitination. To our knowledge, this work is the most comprehensive analysis of a regulatory network of ubiquitination that impinges on acetyltransferase in filamentous pathogens. Moreover, our findings are important because we present the novel roles of the Tom1-Gcn5-Ubp14 circuit in fungal virulence, providing novel possibilities and targets to control fungal diseases.

ClSnRK2.3 negatively regulates watermelon fruit ripening and sugar accumulation.

Watermelon (Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid (ABA) signaling pathway gene ClSnRK2.3 might influence watermelon fruit ripening. However, the molecular mechanisms are unclear. Here, we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA4 . Furthermore, we determined that the pyrophosphate-dependent phosphofructokinase (ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase (ClGA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA4 . Besides that, ClSnRK2.3 phosphorylated homeodomain-leucine zipper protein (ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9'-cis-epoxycarotenoid dioxygenase 3 (ClNCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA4 . Altogether, these findings revealed a novel regulatory mechanism in non-climacteric fruit development and ripening.

Bi-allelic loss-of-function variants in TMEM147 cause moderate to profound intellectual disability with facial dysmorphism and pseudo-Pelger-Huët anomaly

SummaryThe transmembrane protein TMEM147 has a dual function: first at the nuclear envelope, where it anchors lamin B receptor (LBR) to the inner membrane, and second at the endoplasmic reticulum (ER), where it facilitates the translation of nascent polypeptides within the ribosome-bound TMCO1 translocon complex. Through international data sharing, we identified 23 individuals from 15 unrelated families with bi-allelic TMEM147 loss-of-function variants, including splice-site, nonsense, frameshift, and missense variants. These affected children displayed congruent clinical features including coarse facies, developmental delay, intellectual disability, and behavioral problems. In silico structural analyses predicted disruptive consequences of the identified amino acid substitutions on translocon complex assembly and/or function, and in vitro analyses documented accelerated protein degradation via the autophagy-lysosomal-mediated pathway. Furthermore, TMEM147-deficient cells showed CKAP4 (CLIMP-63) and RTN4 (NOGO) upregulation with a concomitant reorientation of the ER, which was also witnessed in primary fibroblast cell culture. LBR mislocalization and nuclear segmentation was observed in primary fibroblast cells. Abnormal nuclear segmentation and chromatin compaction were also observed in approximately 20% of neutrophils, indicating the presence of a pseudo-Pelger-Huët anomaly. Finally, co-expression analysis revealed significant correlation with neurodevelopmental genes in the brain, further supporting a role of TMEM147 in neurodevelopment. Our findings provide clinical, genetic, and functional evidence that bi-allelic loss-of-function variants in TMEM147 cause syndromic intellectual disability due to ER-translocon and nuclear organization dysfunction.

Sensory Characteristics and Microbiological Quality Changes of Nile Tilapia Fillet Processed by Various Sous-vide Conditions During Chilled Storage

Sensory characteristics and microbiological quality of Nile tilapia cooked with various sous-vide (SV) conditions including 50 and 60°C, for 30, 45 and 60 min (S5-30, S5-45, S5-60, S6-30, S6-45 and S6-60) and their changes during storage (4°C) were investigated, compared with control (cooking with boiling water). The result found that increasing temperature and time of SV accelerated protein degradation, both myofibrillar protein and connective tissue. This affected to the lower water-holding capacity (WHC) and shear force of samples treated with severe SV conditions (S6-45 and S6-60) (P&lt;0.05). SV technique may less contribution to the flavor formation, particularly lipid oxidation products, since there were no differences in total volatile base nitrogen (TVB-N) content and thiobarbituric acid reactive substances (TBARS) value among all samples (P&gt;0.05). However, SV cooked samples had higher sensory scores than control, both at day 0 (before storage) or throughout the storage time. This suggested the potential to improve consumer acceptability by this technique. In this study, SV at 60°C, 60 min seem to be the optimal condition for tilapia fillet, which can preserve at 4℃ for at least 6 weeks without any spoilage.

Mutations at the C-terminus of CDC42 cause distinct hematopoietic and autoinflammatory disorders

Pathogenic missense variants in cell division control protein 42 (CDC42) differentially affect protein function, causing a clinically wide phenotypic spectrum variably affecting neurodevelopment, hematopoiesis, and immune response. More recently, 3 variants at the C-terminus of CDC42 were proposed to similarly impact protein function and cause a novel autoinflammatory disorder. We sought to clinically and functionally classify these variants to improve patient management. Comparative analysis of the available clinical data and medical history of patients was performed. Invitro and invivo studies were carried out to functionally characterize individual variants. Differently from what had previously been observed for the p.R186C change causing neonatal-onset cytopenia, autoinflammation, and recurrent hemophagocytic lymphohistiocytosis, p.C188Y and p.∗192Cext∗24 promoted accelerated protein degradation. Unprenylated CDC42C188Y did not behave as a membrane-bound protein, whereas the residual CDC42∗192Cext∗24 mutant replicated the CDC42R186C behavior, being targeted to the Golgi apparatus in a palmitoylation-dependent manner. Assessment of invitro polarized migration and development in Caenorhabditis elegans documented a loss-of-function behavior of the p.C188Y and p.∗192Cext∗24 variants. Consistently, the 3 pathogenic variants were associated with different clinical presentations, with dysmorphisms, severity, and age of onset of cytopenia and extent of autoinflammation representing major differences. Pathogenic variants at the CDC42 C-terminus differently impact protein stability, localization, and function, and cause different diseases, with p.R186C specifically associated with neonatal-onset pancytopenia and severe autoinflammation/hemophagocytic lymphohistiocytosis requiring emapalumab and bone marrow transplantation, and p.C188Y and p.∗192Cext∗24 causing anakinra-sensitive autoinflammation.

Metabolic changes contributing to large biomass production in the Arabidopsis ppGpp-accumulating mutant under nitrogen deficiency

The Arabidopsis ppGpp-overproducing mutant indicates a larger biomass than wild type by modulated amino-acid metabolism under nitrogen-limiting conditions. The regulatory nucleotide, guanosine 3', 5'-bis(pyrophosphate; ppGpp)-originally identified in Escherichia coli-controls gene expression and enzyme activities in the bacteria and plastids of plant cells. We recently reported that the ppGpp over-producing mutant of Arabidopsis thaliana had a larger shoot weight than wild type (WT), especially under nutrient-deficient conditions. However, the mechanisms behind the influence of ppGpp on plant growth and biomass remain elusive. To understand the impact of the ppGpp accumulation on plant growth, we characterized metabolic changes in the ppGpp-overproducing mutant upon transition from nitrogen-rich to nitrogen-limiting concentrations. We found that the fresh weight of the mutant was significantly larger than WT when the total nitrogen source (KNO3 and NH4NO3) concentration was below 0.9mM. When the nitrogen content in the medium decreased, aromatic and branched-chain amino acids increased in WT due to accelerated protein degradation and/or attenuated protein synthesis. These amino-acid levels in the ppGpp over-accumulating mutant decreased upon nitrogen deficiency. The results suggest that the ppGpp-overaccumulation affects amino-acid and protein homeostasis and facilitates growth under nitrogen-limiting conditions.

A bis-pyridinium fullerene derivative induces apoptosis through the generation of ROS in BCR-ABL-positive leukemia cells

A fusion protein, Breakpoint cluster region-Abelson (BCR-ABL) is responsible for the development of chronic myeloid leukemia (CML) and acute lymphocytic leukemia (ALL). Inhibitors against BCR-ABL are effective for the treatment of leukemia; however, a gatekeeper mutation (T315I) in BCR-ABL results in resistance to these inhibitors, which markedly impedes their efficacy. We herein demonstrated that a bis-pyridinium fullerene derivative (BPF) significantly induced apoptosis in human CML-derived K562 cells and ALL-derived SUP-B15 cells via the generation of reactive oxygen species (ROS). BPF reduced the expression of Bcr-Abl mRNA by inhibiting expression of c-Myc through ROS production. BPF also accelerated protein degradation of BCR-ABL through ROS production. Furthermore, BPF down-regulated the expression of not only BCR-ABL but also T315I-mutated BCR-ABL in ROS-dependent manner. As a result, BPF effectively induced apoptosis in transformed Ba/F3 cells expressing both BCR-ABL and T315I-mutated BCR-ABL. Collectively, these results indicate the potential of BPF as an effective leukemia drug that overcomes resistance to BCR-ABL inhibitors.

Reduction of biogenic amines accumulation with improved flavor of low-salt fermented bream (Parabramis pekinensis) by two-stage fermentation with different temperature

How to ensure low-salt fermented food safety with good flavor under non-acidic conditions is a challenge. Temperature is one of the key environmental parameters that affect the growth of microorganism and enzyme activity. The effects of fermentation for 4 days at 4 °C, 10 °C, 20 °C and 20-10 °C (20 °C for 2 days followed by 10 °C for 2 days) on the flavor, microbial and physicochemical properties of low-salt fermented bream were investigated. Results showed that compared with 4 °C-fermentation, higher fermentation temperature (10 °C, 20 °C, 20-10 °C) could accelerate protein degradation and facilitate the formation of flavor substances during fermentation of bream. The volatile flavor compounds of 20 °C and 20-10 °C two-stage fermentation groups were similar. After 4 days of fermentation, highest content of alcohols, ketones and esters were observed in two-stage fermentation group (20-10 °C). For comparison with 20 °C-fermentation, two-stage fermentation (20-10 °C) reduced the contents of total volatile basic nitrogen, cadaverine, putrescine and total biogenic amines significantly (p < 0.05), and inhibited the growth of harmful microorganisms (Pseudomonas and Coliforms). Results suggested that two-stage fermentation with different temperature provided a potential method that facilitated improving the flavor and safety of low-salt fermented bream.

The impact of Flavourzyme on flavor, antioxidant activity, and sensory attributes of salted grass carp by brine injection and brining

Enzyme injection is vital for improving the sensory attributes and processing characteristics of meat products by enhancing proteolysis. However, studies regarding the appropriate dose addition for accelerating protein degradation in grass carp are minimal. This study aimed to investigate the impact of Flavourzyme® on the flavor quality and antioxidant activity of salted grass carp via brine injection and brining. Flavourzyme was added at doses of 0, 5, 10, 20, and 30 leucine aminopeptidase units (LAPU) per kilogram of raw meat. The results indicated that adding Flavourzyme promoted proteolysis, which was reflected by the enhanced total free amino acid content (from 3.7414 g kg-1 to 4.9160 g kg-1 in the brining group and from 3.8039 g kg-1 to 5.4061 g kg-1 in the injection group) and a decrease in salt soluble and insoluble protein (P < 0.05). The antioxidant activity was improved, and the thiobarbituric acid reactive substance value in salted carp decreased due to the higher content of the protein hydrolysis product (P < 0.05). All sensory attributes were improved significantly, especially when using brine injection (P < 0.05). Brine injection was helpful to diffuse the Flavourzyme, resulting in stronger proteolysis. The appropriate Flavourzyme dose was 10 LAPU kg-1 in the injection group and 20 LAPU kg-1 in the brining group. Therefore, moderate Flavourzyme addition was excellent in improving sensory attributes and storage characteristics, whereas injection represented a novel method to obtain a similar fish meat quality in a shorter time and with less added Flavourzyme. © 2021 Society of Chemical Industry.

Respective, Time‐dependent Phosphorylation Modules Shaping Phosphoproteome Abundance and Turnover

Protein phosphorylation mediates protein-protein interactions and alters protein structure, stability, and subcellular localization—frequently in a modification site-specific manner. However, how phosphorylation impacts protein lifetime remains to be illustrated. We have devised a proteomic method, DeltaSILAC, to quantitatively assess the influence of site-specific phosphorylation on the turnover of thousands of proteins in live cells (1). We discovered many phosphosites prolonging protein lifetime, challenging the previous notion that phosphorylation mostly accelerates protein degradation. Extending this research analyzing steady-state cells, we herein aimed to characterize how the timing and duration of phosphorylation signaling dedicate cell phenotypes. In a classic signaling system, the differential stimulation of epidermal and nerve growth factors (EGF and NGF) can determine the cell fate. Whereas EGF “transiently” stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation, NGF stimulation induces “sustained” activation of MAPKs and cell differentiation. Herein, we applied both DeltaSILAC and label-free phosphoproteomics. We developed DeltaSILAC with a pulse labeling using stable isotope-labeled amino acids in cells (pSILAC) accompanying the EGF and NGF stimulations on PC12 cells. Our time-course design embraced both short- and long-term points: 0-sec, 15-sec, 30-sec, 1-min, 2-min, 5-min, 10-min, 30-min, 1-hour, 2 hour, 4-hour, 8-hour, 12-hour, 24-hour, 48-hour, and 72-hour. We also generated matching protein- and mRNA- profiles after 30 mins following stimulations, so that phosphorylation regulations can be dissected from gene expression dynamics. We utilized a novel peptide-level matching algorithm so that both abundance and lifetime can be measured and resolved for each phosphosite. By using a reproducible mass spectrometry method named data-independent acquisition (DIA-MS), collectively, we quantified 40,000 phosphopeptides and 9,000 proteins throughout the experiment, both globally recapitulated the “transient” and “sustained” patterns of EGF and NGF signaling. We discovered specific tyrosine phosphorylation as early as 15-30 seconds. Interesting EGFR phosphosites activated by NGF treatment were ascribed to P38 kinase activity. The modeling of our unique time-series datasets of long-term time points indicates that phosphosites involved in EGF/NGF signaling transduction and mRNA processing shortened the corresponding protein lifetime, whereas phosphosites in the apoptotic process stabilized protein expression. We conclude that while early phosphorylation events initiate signaling functions through phosphate-dependent transfer, the longer-lasting phosphorylation events could shape proteome stability and proteostasis in cell fate determination.1. C. Wu et al., Global and Site-Specific Effect of Phosphorylation on Protein Turnover . Developmental cell 10.1016/j.devcel.2020.10.025 (2020).

Adipose tissue lipolysis is regulated by PAQR11 via altering protein stability of phosphodiesterase 4D

Fat storage and mobilization in adipose tissue play a central role in energy metabolism and are directly linked to the development of obesity. Upon starvation, fat is mobilized from adipose tissue by lipolysis, a process by which triglycerides are hydrolyzed to free fatty acids to be used as an energy source in skeletal muscles and other tissues. However, how lipolysis is activated by starvation is not fully known. In this study, we demonstrate that PAQR11, a member of the progesterone and AdipoQ receptor family, regulates starvation-mediated lipolysis. Paqr11-deleted mice are resistant to high-fat diet-induced obesity. Paqr11 deletion promotes lipolysis in white adipose tissue, characterized by increased phosphorylations of hormone-sensitive lipase (HSL) and perilipin 1 (PLIN1) and elevated serum levels of glycerol and free fatty acids. PKA activity and cAMP levels in white adipose tissue are also increased by Paqr11 deletion, accompanied by accelerated protein degradation of phosphodiesterase 4D (PDE4D). Mechanistically, PAQR11 decreases the interaction of PDE4D with SKP1-CUL1-FBXO2 E3 ligase complex, thus modulating the polyubiquitination/degradation of PDE4D. Fasting decreases the expression of the Paqr11 gene, and starvation-induced lipolysis in white adipose tissue is enhanced by Paqr11 deletion, while insulin-mediated suppression of lipolysis is not affected. Collectively, these results reveal that PAQR11 regulates lipolysis of adipose tissue and affects high-fat diet-induced obesity.

Geniposide improves insulin resistance through AMPK-mediated Txnip protein degradation in 3T3-L1 adipocytes.

Thioredoxin-interacting protein (Txnip) has emerged as a key regulator of insulin resistance. In this study, we investigated the roles of geniposide and Txnip in insulin resistance in differentiated 3T3-L1 adipocytes. Our results revealed that geniposide markedly enhanced glucose uptake, increased the protein levels of insulin receptor substrate (IRS)-1 and GLUT-1, and prevented the phosphorylation of IRS-1 and Akt Thr308 induced by insulin resistance in 3T3-L1 adipocytes. We also observed that geniposide accelerated protein degradation of Txnip through proteasome pathway, and knockdown of Txnip with small interfering RNA attenuated the effect of geniposide on insulin signaling molecules, implying that Txnip played a pivotal role in the regulation of insulin signaling molecules by geniposide in 3T3-L1 adipocytes. Furthermore, geniposide induced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in the presence of high glucose in differentiated 3T3-L1 adipocytes, while compound C, an inhibitor of AMPK, prevented the effect of geniposide on Txnip degradation and the regulation of glucose uptake and insulin signaling molecules including p-IRS-1, IRS-1, and GLUT-1 in differentiated 3T3-L1 adipocytes. Taken together, all these findings suggest that geniposide improves the insulin signaling defect possibly by AMPK-mediated Txnip degradation in 3T3-L1 adipocytes.

Examining Targeted Protein Degradation from Physiological and Analytical Perspectives: Enabling Translation between Cells and Subjects.

The ability to target specific proteins for degradation may open a new door toward developing therapeutics. Although effort in chemistry is essential for advancing this modality, i.e., one needs to generate proteolysis targeting chimeras (bifunctional molecules, also referred to as PROTACS) or "molecular glues" to accelerate protein degradation, we suspect that investigations could also benefit by directing attention toward physiological regulation surrounding protein homeostasis, including the methods that can be used to examine changes in protein kinetics. This perspective will first consider some metabolic scenarios that might be of importance when one aims to change protein abundance by increasing protein degradation. Specifically, could protein turnover impact the apparent outcome? We will then outline how to study protein dynamics by coupling stable isotope tracer methods with mass spectrometry-based detection; since the experimental conditions could have a dramatic effect on protein turnover, special attention is directed toward the application of methods for quantifying protein kinetics using in vitro and in vivo models. Our goal is to present key concepts that should enable mechanistically informed studies which test targeted protein degradation strategies.